Eph Receptor Family Research Articles

Ephrin‐B3 Binds Specifically to B Lymphocytes in Blood and Induces Migration

Eph receptors and ephrin ligands have been shown to be differentially expressed on leucocytes. Here, we show that one member of the ephrin-B subfamily of ephrins, ephrin-B3, specifically binds to B lymphocytes in blood. No binding was observed to T lymphocytes or monocytes. The ephrin-B3 binding receptor on B lymphocytes is so far not identified, but our results here indicate that ephrin-B3 binds to a protein not belonging to the Eph receptor family. Recently, we have shown that ephrin-B3 binds to a sulphated cell surface receptor on HEK293T cells and that this binding can be blocked with heparin. Ephrin-B3 binding to B lymphocytes is partially affected by heparin, and a basic amino acid in the extracellular juxtamembrane region, Arg-188, is here shown to be involved in this binding. The functional consequence of ephrin-B3 binding to B lymphocytes is induced migration, in particular of the memory cells. To conclude, ephrin-B3 binds to B lymphocytes, most likely via a non-classical receptor, and induces migration of the memory B cell subpopulation.

Expression Profiling of the Ephrin (EFN) and Eph Receptor (EPH) Family of Genes in Atherosclerosis-Related Human Cells

Ephrin B1 and its cognate receptor, Eph receptor B2, key regulators of embryogenesis, are expressed in human atherosclerotic plaque and inhibit adult human monocyte chemotaxis. Few data exist, however, regarding the gene expression profiles of the ephrin (EFN) and Eph receptor (EPH) family of genes in atherosclerosis-related human cells. Gene expression profiles were determined of all 21 members of this gene family in atherosclerosis-related cells by reverse transcription-polymerase chain reaction analysis. The following 17 members were detected in adult human peripheral blood monocytes: EFNA1 and EFNA3 - EFNA5 (coding for ephrins A1 and A3 - A5); EPHA1, EPHA2, EPHA4 - EPHA6 and EPHA8 (coding for Eph receptors A1, A2, A4 - A6 and A8); EFNB1 and EFNB2 (coding for ephrins B1 and B2); and EPHB1 - EPHB4 and EPHB6 (coding for Eph receptors B1 - B4 and B6). THP-1 monocytic cells, Jurkat T cells and adult arterial endothelial cells also expressed multiple EFN and EPH genes. These results indicate that a wide variety of ephrins and Eph receptors might affect monocyte chemotaxis, contributing to the development of atherosclerosis. Their pathological significance requires further study.

Lithocholic Acid Is an Eph-ephrin Ligand Interfering with Eph-kinase Activation

Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or de-regulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumour phenotype. In particular EphA2 upregulation is correlated with tumour stage and progression and the expression of EphA2 in non-trasformed cells induces malignant transformation and confers tumorigenic potential. Based on these evidences our aim was to identify small molecules able to modulate EphA2-ephrinA1 activity through an ELISA-based binding screening. We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 interaction (Ki = 49 µM). Since each ephrin binds many Eph receptors, also LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Structurally related bile acids neither inhibited Eph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibited EphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate and colon adenocarcinoma cell lines (IC50 = 48 and 66 µM, respectively) without affecting cell viability or other receptor tyrosine-kinase (EGFR, VEGFR, IGFR1β, IRKβ) activity. LCA did not inhibit the enzymatic kinase activity of EphA2 at 100 µM (LANCE method) confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells. In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, as ephrin signalling is a key player in the intestinal cell renewal, our work could provide an interesting starting point for further investigations about the role of LCA in the intestinal homeostasis.

Copy Number Variations of EphA3 Are Associated With Multiple Types of Hematologic Malignancies

EphA3 is a component of the Eph receptor family, the largest subgroup of the receptor tyrosine kinase (RTK) family. A recent array-based study implicated the presence of copy-number variations (CNVs) of EphA3 in the genomes of acute myelogenous leukemia. CNVs are present in the general population at varying degrees, and have been found to associate with various types of diseases including hematologic malignancies. However, most of the current studies focused on the genome-wide screening of CNVs, and the functional impact of such regions needs to be extensively investigated in large number of clinical samples. In our study, we collected 617 bone marrow samples from multiple types of hematologic malignancies as well as healthy controls. DNA copy numbers and mRNA levels of EphA3 in these samples were examined. We found significant association between the CNVs of EphA3 and these hematologic malignancies including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), and myelodysplastic syndrome (MDS). We also observed a positive correlation between the relative mRNA level and gene dosage of EphA3. The CNVs of EphA3 were associated with multiple types of hematologic malignancies including ALL, AML, CLL, CML, MM, and MDS.

The EphB4 receptor promotes the growth of melanoma cells expressing the ephrin‐B2 ligand

The EphB4 receptor promotes the growth of melanoma cells expressing the ephrin‐B2 ligand

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What is the difference between reading an online versus a print issue of PCMR? Reading an article targeted by a keyword search online, you may miss the wealth of information provided for the pigment and melanoma communities and will likely not benefit from recently added features that make PCMR a more rounded and mature journal. Those sections include a timely summary of recently published studies selected by the editors based on importance and relevance to our readers (News and Views, N&V), discussion forums (Hypothesis, Perspectives, Commentaries, Letter To The Editor), a reagent database (Resource), and a feature highlighting select members of our communities (Profile). You could read these sections online, but chances are that you will not move forward and backwards through online articles, as you might flip through pages of a print issue. Here I bring to your attention the ‘perks’ we now provide, with the hope that you will catch up on some thought-provoking reading. This issue includes three N&V on topics you may not have anticipated but will surely appreciate. The first covers the newly discovered concept of MicroRNAs (miRs) being ‘fooled’ by pseudogenes (working as decoys). Those genomic sequences that have been believed to lack function are now demonstrated to ‘hijack’ miRs and therefore attenuate their function. Based on evidence regarding Pten and Ras pseudogenes and recent discoveries establishing the link between MITF and DICER (a key component of the miR processing machinery; see N&V in a recent PCMR issue, Vol 23 p. 483), the importance and relevance of these observations for melanoma or melanocyte function are easy to grasp. Another N&V in this issue discusses the newly discovered link between hypoxia and control of TRYP-2 (tyrosinase-related protein 2 aka DCT), which was recently revealed in worms. Notably, DCT induced by hypoxia inducible factor (HIF) in neurons, was secreted and taken up by germ cells where it inhibited apoptosis, thereby highlighting novel mechanism for production and remote function of TYRP-2 by oxygen tension. TYRP-2 protection from apoptosis was confirmed in melanoma cells, further highlighting its relevance to our readers. A third N&V discusses the recent discovery that MAPK and AKT signaling pathways regulate protein translation in cancer cells by converging on a translational inhibitor eIF4E-bnding protein 1 (4E-BP1). Would 4E-BP1 serve as a good target for therapy, given its control by MAPK and AKT signaling, which are often deregulated in melanoma? The past few months have brought a wealth of new ideas regarding melanoma therapy. A new generation of B-Raf inhibitors and the initial indication of success for CTL4A-based immunotherapy offer exciting opportunities for treatment of this devastating disease, as discussed in a N&V published herein. Nonetheless, the search for new targets and novel approaches continues. Two studies published in this issue point to the possible exploitation of ER stress components in melanoma therapy. Given that ER stress is a major constituent of cellular responses to misfolded proteins and is commonly observed following treatment with drugs perturbing normal cellular homeostasis, exploiting ER stress components may offer new therapeutic modalities. Fundamental to our understanding of melanoma biology is the role of related stem cells. A commentary in a recent issue of PCMR by Dennis Roop and colleagues (Vol 23 p. 517) provided a timely overview of skin stem cells. Yet, some basic principles of melanoma stem cells are not yet settled – for one – we have not reached a consensus as to whether they exist as a genetically defined population. In a Letter to the Editor, Mark Shackleton sheds new light on technical nuances that may account for different results and conclusions reported by different groups who have been studying melanoma stem cells. Understanding these issues puts us in a better position to evaluate results of these studies and consider the next line of experiments. Notably, the founder of the melanocyte stem cell field, Dr. Shin-ichi Nishikawa, is the subject of our Profile section in this issue, which describes how his discovery of stem cells came about. Our contributors continue to address fundamental mechanistic questions in melanoma biology. Two studies in this issue present new insight into mechanisms underlying tumor microenvironment in melanoma progression. Pasquale and her colleagues reveal the importance of new components of the Eph receptor family, namely EphB4 in growth of melanoma cells expressing the ephrin-B2 ligand. The effect of EphB4 on blood vessels enlargement is presented on the cover of this issue. In a separate study, Indra and his colleagues demonstrate how reduced expression of RxRα in keratinocytes increased melanoma metastasis. Notably, this report can not only be read, but also viewed online, in a PubCast at SciVee (http://www.scivee.tv/node/17730). Flip the page or Scroll online – it is up to you to choose how you benefit from the comprehensive coverage available in PCMR. Whether you scroll or flip – enjoy and cite!

Ephexin4 and EphA2 mediate cell migration through a RhoG-dependent mechanism

EphA2, a member of the Eph receptor family, is frequently overexpressed in a variety of human cancers, including breast cancers, and promotes cancer cell motility and invasion independently of its ligand ephrin stimulation. In this study, we identify Ephexin4 as a guanine nucleotide exchange factor (GEF) for RhoG that interacts with EphA2 in breast cancer cells, and knockdown and rescue experiments show that Ephexin4 acts downstream of EphA2 to promote ligand-independent breast cancer cell migration and invasion toward epidermal growth factor through activation of RhoG. The activation of RhoG recruits its effector ELMO2 and a Rac GEF Dock4 to form a complex with EphA2 at the tips of cortactin-rich protrusions in migrating breast cancer cells. In addition, the Dock4-mediated Rac activation is required for breast cancer cell migration. Our findings reveal a novel link between EphA2 and Rac activation that contributes to the cell motility and invasiveness of breast cancer cells.

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.

Abstract 5216: An Akt-EphA2 reciprocal regulatory loop controls tumor cell migration and invasion

Abstract EphA2 is a member of Eph family receptor tyrosine kinases that has been implicated in many types of human cancers. Perplexingly, both pro- and anti-oncogenic properties have been attributed to EphA2 kinase. We report here that a possible cause for this apparent paradox is diametrically opposite roles of EphA2 in regulating cell migration and invasion. While activation of EphA2 with its ligand ephrin-A1 inhibited chemotactic migration of glioma and prostate cancer cells, EphA2 overexpression promoted migration in a ligand-independent manner. Surprisingly, the latter effects required phosphorylation of EphA2 on serine 897 by Akt, and S897A mutation abolished ligand-independent promotion of cell motility. Ephrin-A1 stimulation of EphA2 negated Akt activation by growth factors and caused EphA2 dephosphorylation on S897. In human astrocytoma, S897 phosphorylation was correlated with tumor grades and Akt activation, suggesting that the Akt-EphA2 crosstalk may contribute to brain tumor progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5216.

Abstract 3951: EphA10 exhibits anti-apoptotic activity through its dominant-negative role for EphA signaling in cancer cells

Abstract Eph receptors are members of receptor tyrosine kinase family. They are activated by binding of their ligands, ephrins, which regulate numerous physiological responses, including axon guidance, and pattern formation during development. In addition to their important roles during development, several members of the Eph receptor family have been reported to function in carcinogenesis. Recently EphA10 was isolated as the 16th member of the Eph receptor family. EphA10 is expressed in normal testis and binds to ephrin-A. However, biological functions of EphA10 remain unclear. We demonstrate here that EphA10 functions as a dominant-negative receptor for EphA signaling. Our results show that EphA10 is over-expressed in cancer tissues, especially breast and lung cancer tissues as compared with patient-matched adjacent normal tissues. EphA10 binds to ephrin-A2, 3, 4, and 5. Analysis of the amino acid sequence of EphA10 revealed that EphA10 does not have Lys and Asp in the kinase domain, which are necessary for kinase activity nor two Tyr in the juxtamembrane domain, which are necessary for phosphorylation. As expected from the amino acid sequence, no intrinsic tyrosine kinase activity of EphA10 was detected despite its interaction with ephrin-A. To further investigate the mechanism of EphA10 signaling, we tested the role of EphA10 in modulating ephrin-A4 activity. In our experiment, ephrin-A4 suppressed phosphorylation of ERK1/2 in HEK293 cells. EphA10 over-expression in HEK293 cells reduced the effect of ephrin-A4 on ERK1/2. Conversely, reduction of EphA10 expression by antisense oligonucleotides induced apoptosis in the breast cancer cell line ZR-75-1. In addition to its role in shunting ephrin-A response through binding to ephrin-A directly, we found that EphA10 formed a hetero-complex with functional EphA7, suggesting that EphA10 may block EphA signaling by forming non-functional hetero complexes with other functional EphA receptors. Our studies demonstrated that EphA10 has anti-apoptotic function in cancer cells by blocking EphA signaling through its dominant-negative activity. Thus, EphA10 potentially plays an important role in cancer cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3951.

Ephrin-B2 regulates endothelial cell morphology and motility independently of Eph-receptor binding

The transmembrane protein ephrin-B2 regulates angiogenesis, i.e. the formation of new blood vessels through endothelial sprouting, proliferation and remodeling processes. In addition to essential roles in the embryonic vasculature, ephrin-B2 expression is upregulated in the adult at sites of neovascularization, such as tumors and wounds. Ephrins are known to bind Eph receptor family tyrosine kinases on neighboring cells and trigger bidirectional signal transduction downstream of both interacting molecules. Here we show that ephrin-B2 dynamically modulates the motility and cellular morphology of isolated endothelial cells. Even in the absence of Eph-receptor binding, ephrin-B2 stimulates repeated cycling between actomyosin-dependent cell contraction and spreading episodes, which requires the presence of the C-terminal PDZ motif. Our results show that ephrin-B2 is a potent regulator of endothelial cell behavior, and indicate that the control of cell migration and angiogenesis by ephrins might involve both receptor-dependent and receptor-independent activities.

Ephrin-B2 regulates endothelial cell morphology and motility independently of Eph-receptor binding

The transmembrane protein ephrin-B2 regulates angiogenesis, i.e. the formation of new blood vessels through endothelial sprouting, proliferation and remodeling processes. In addition to essential roles in the embryonic vasculature, ephrin-B2 expression is upregulated in the adult at sites of neovascularization, such as tumors and wounds. Ephrins are known to bind Eph receptor family tyrosine kinases on neighboring cells and trigger bidirectional signal transduction downstream of both interacting molecules. Here we show that ephrin-B2 dynamically modulates the motility and cellular morphology of isolated endothelial cells. Even in the absence of Eph-receptor binding, ephrin-B2 stimulates repeated cycling between actomyosin-dependent cell contraction and spreading episodes, which requires the presence of the C-terminal PDZ motif. Our results show that ephrin-B2 is a potent regulator of endothelial cell behavior, and indicate that the control of cell migration and angiogenesis by ephrins might involve both receptor-dependent and receptor-independent activities.

EphA1 receptor silencing by small interfering RNA has antiangiogenic and antitumor efficacy in hepatocellular carcinoma

The Eph family of receptor tyrosine kinases has emerged as one of the pivotal regulators of tumor angiogenesis. EphA1, the first identified member of the Eph receptor family, has been found to be overexpressed in several types of human tumors. A recent report indicated that EphA1 was overexpressed in hepatocellular carcinoma (HCC) and that elevated expression of EphA1 can promote proliferation of HCC cells through stimulation by exogenous Ephrin-A1. To investigate the role of EphA1 in angiogenesis and progression of HCC, we down-regulated EphA1 by RNA interference (RNAi) technology, in an HCC-derived cell line with a high level of EphA1 expression. We established a stable knockdown clone named SiEphA1/Huh-7. The knockdown resulted in decreased proliferation of Huh-7 cells, as well as decreased motility and invasion capability in vitro. siRNA-based EphA1 knockdown also down-regulated the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2 and -9. Interestingly, the suppression of EphA1 expression in Huh-7 cells reduced their outgrowth when inoculated in the subcutaneous space in the flank of nude mice, presumably through angiogenesis inhibition since microvessel density was found to be inhibited.

Eph-B2 Receptor Tyrosine Kinase Is Overexpressed in Waldenstrom's Macroglubulinemia and Plays a Major Role in Its Interaction with the Bone Marrow Microenvironment.

Abstract 2935Poster Board II-911 INTRODUCTION:Waldenstrom's macroglobulinemia (WM) is a low-grade lymphoma characterized by the presence of lymphoplasmacytic lymphoma cells in the bone marrow (BM). The BM microenvironment was shown to promote growth and proliferation of WM cells. The Eph receptor family are receptor tyrosine kinases RTKs activated by ephrin which a cell-surface protein, and the interaction between the receptor on one cell and the ligand on other cells promotes the activity of the receptor. Eph receptors are known to control processes such as cell growth, proliferation, migration, and invasion, and their expression level was shown to be elevated in a wide range of solid tumors. However, their role in WM was never explored. METHODS AND RESULTS:Using phosphor-RTK array kit, we have screened the activity of 42 RTKs in CD19+ cells from 4 WM patients, WM cell line BCWM1, IgM secreting cell lines MEC1 and RL, and CD19+ cells from the BM of 4 healthy donors. We found that one of the most significant RTKs which showed high activation in all WM cells from patient sample and cells, compared to all normal cells was Eph-B2 receptor. We have used ephrin-B2, the ligand of the receptor Eph-B2, to test the signaling pathways involved in the activity of this receptor. We found, by immunoblotting, that ephrin-B2 induce phosphorylation of the receptor in a bell-curve manner, with the activity peaking in 100 ng/ml, while the expression of the total form of the receptor was unchanged over arrange of 0-1000 ng/ml of ephrin. A comparable activation was found for several cell-adhesion related proteins including FAK, SRC, paxillin, P130 and cofillin. These finding indicated a major role of the Eph-B2 receptor cell adhesion of WM cell. We have coated plates with increasing concentration of ephrin-B2 and tested the adhesion of BCWM1, MEC1 and RL to ephrin. Again, we found the adhesion peaked at the concentration of 100nM of ephrin. In contrast, ephrin-B2 had no chemotactic effect on WM cells. BM microenvironment components including BM stromal cells (BMSCs) and endothelial cells were shown to enhance the proliferation of WM cells detected by thymidine uptake assay. Ephrin-B2 was shown to be expressed on both endothelial cells and BMSCs. The inhibition of either the ephrin-B2 on endothelial cells and BMSCs, or inhibition of the Eph-B2 receptor on WM cell reduced the adhesion of WM cell to both endothelial cells and BMSCs and decreased the increase of WM cell proliferation induced by endothelial cells and BMSCs. The combination of both inhibition ephrin-B2 and Eph-B2 did not have an additive effect compared to each of them alone. Moreover, the inhibition of either ephrin-B2 or Eph-B2 reduced the activation of cell adhesion-related proteins. CONCLUSION:In conclusion, we showed that the Eph-B2/ephrin-B2 axis was activated in WM cells and that it is important for the adhesion and proliferation of WM cells induced by the BM microenvironment. These findings provide a novel therapeutic target for WM. Disclosures:Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Biological significance of EPHA2 expression in neuroblastoma

Neuroblastoma is a pediatric solid tumor that exhibits striking clinical bipolarity. Despite extensive efforts to treat unfavorable neuroblastoma, survival rate of children with the disease is among the lowest. Previous studies suggest that EPHA2, a member of the EPH family receptor kinases, can either promote or suppress cancer cell growth depending on cellular contexts. In this study, we investigated the biological significance of EPHA2 in neuroblastoma. It was found that tumorigenic N-type neuroblastoma cell lines expressed low levels of EPHA2, whereas hypo-tumorigenic S-type neuroblastoma cell lines expressed high levels of EPHA2 (p<0.005). Notably, inhibitors of DNA methylation and histone deacetylase enhanced EPHA2 expression in N-type cells, suggesting that EPHA2 is epigenetically silenced in unfavorable neuroblastoma cells. Furthermore, ectopic high-level expression of EPHA2 in N-type neuroblastoma cell lines resulted in significant growth suppression. However, Kaplan-Meier survival analysis showed that high EPHA2 expression was not associated with a good disease outcome of neuroblastoma, indicating that EPHA2 is not a favorable neuroblastoma gene, but a growth suppressive gene for neuroblastoma. Accordingly, EPHA2 expression was markedly augmented in vitro in neuroblastoma cells treated with doxorubicin, which is commonly used for treating unfavorable neuroblastoma. Taken together, EPHA2 is one of the effectors of chemotherapeutic agents (e.g., gene silencing inhibitors and DNA damaging agents). EPHA2 expression may thus serve as a biomarker of drug responsiveness for neuroblastoma during the course of chemotherapy. In addition, pharmaceutical enhancement of EPHA2 by non-cytotoxic agents may offer an effective therapeutic approach in the treatment of children with unfavorable neuroblastoma.

Eph Receptor Tyrosine Kinases and Ephrin Ligands Are Epigenetically Inactivated in Acute Lymphoblastic Leukemia and Are Potential New Tumor Suppressor Genes in Human Leukemia.

The Eph (erythroprotein-producing hepatoma amplified sequence) family receptor tyrosine kinases and their ephrin ligands (ephrins) are involved in a variety of functions in normal cell development and cancer. We have identified several members of this family as potential targets of aberrant DNA methylation using Methylated CpG Island Amplification (MCA) / DNA promoter microarray technology. This is of importance as there are no prior reports of potential Eph receptor or Ephrin epigenetic inactivation in human leukemia. To further investigate the role of Eph receptor and ephrin family genes in leukemia, we have analyzed their DNA methylation status in a panel of 23 leukemia cell lines and 65 primary ALL patient samples. Aberrant DNA methylation of 9 of these genes (EPHA4, EPHA5, EPHA6, EPHB2, EPHB3, EPHB4, EphrinA5, Ephrin B2, and EphrinB3) was detected in multiple leukemia cell lines but not in normal samples by bisulfite pyrosequencing. In ALL patient samples, the frequencies of DNA methylation detected in the promoter regions of these genes ranged from 23% to 87% for EPHA4, EPHA5, EPHA6, EPHB2, EPHB3, EPHB4, EphrinA5, Ephrin B2, and EphrinB3. Expression analysis of 3 of these genes (EPHA5, EPHB4 and Ephrin B2) in leukemia cell lines by real-time PCR further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with DNA methyltransferase inhibitor 5′-aza-2′-deoxycytidine resulted in gene re-expression. Forced overexpression of EPHB4 using a lentivirus transduction system in Raji cell lines resulted in decreased cell proliferation and adhesion-independent cell growth, as well as in an increase in staurosporine induction of apoptosis. In addition, EPHB4 overexpression resulted in a significant downregulation of phosphorylated Akt pathway but had no effect on mitogen-activated protein kinase pathway. In summary, we describe for the first time the epigenetic suppression of Ephrin receptors and their ligands in human leukemia, indicating that these genes may be potential tumor suppressors in leukemia. Targeting of these pathways may result in the development of new potential therapies and biomarkers for patients with ALL.

The Eph receptor family

The Eph receptor family

Regulation of Ephexin1, a Guanine Nucleotide Exchange Factor of Rho Family GTPases, by Fibroblast Growth Factor Receptor-mediated Tyrosine Phosphorylation

Fibroblast growth factor (FGF) signal is implicated in not only cell proliferation, but cell migration and morphological changes. Several different Rho family GTPases downstream of the Ras/ERK pathway are postulated to mediate the latter functions. However, none have been recognized to be directly coupled to FGF receptors (FGFRs). We have previously reported that EphA4 and FGFRs hetero-oligomerize through their cytoplasmic domains, trans-activate each other, and transduce a signal for cell proliferation through a docking protein, FRS2alpha (Yokote, H., Fujita, K., Jing, X., Sawada, T., Liang, S., Yao, L., Yan, X., Zhang, Y., Schlessinger, J., and Sakaguchi, K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 18866-18871). Here, we have found that ephexin1, a guanine nucleotide exchange factor for Rho family GTPases, constitutes another downstream component of the receptor complex. Ephexin1 directly binds to the kinase domain of FGFR mainly through its DH and PH domains. The binding appears to become weaker and limited to the DH domain when FGFRs become activated. FGFR-mediated phosphorylation of ephexin1 enhances the guanine nucleotide exchange activity toward RhoA without affecting the activity to Rac1 or Cdc42. The FGFR-mediated tyrosine phosphorylation includes, but is not limited to, the residue (Tyr-87) phosphorylated by Src family kinase, which is known to be activated following EphA4 activation. The Tyr-to-Asp mutations that mimic the tyrosine phosphorylation in some of the putative FGFR-mediated phosphorylation sites increase the nucleotide exchange activity for RhoA without changing the activity for Rac1 or Cdc42. From these results, we conclude that ephexin1 is located immediately downstream of the EphA4-FGFR complex and the function is altered by the FGFR-mediated tyrosine phosphorylation at multiple sites.

Phosphorylation of Ephrin-B1 Regulates Dissemination of Gastric Scirrhous Carcinoma

Interaction of the Eph family of receptor protein tyrosine kinase and its ligand ephrin family induces bidirectional signaling via cell-cell contacts. High expression of B-type ephrin is frequently found in various cancer cells, and their expression levels are associated with high invasion of tumors and poor prognosis. However, whether ephrin-B1 actually promotes invasion of cancer cells in vivo has not been shown. We investigated the involvement of ephrin-B1 in regulating the invasiveness of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Reduction of ephrin-B1 expression by short inter-fering RNA or overexpression of phosphorylation-defective mutant suppressed migration and invasion of scirrhous gastric cancer cells in vitro without affecting tumor cell proliferation and apoptosis. Blocking of tyrosine phosphorylation of ephrin-B1 attenuates not only dissemination of cancer cells injected intraperitoneally but also local invasion and dissemination of orthotopically implanted cancer cells in the gastric wall of nude mice. Furthermore, blocking of ephrin-B1 phosphorylation attenuated the activation of Rac1 GTPase in these invasive gastric cancer cells. Our results suggest that tyrosine phosphorylation of ephrin-B1 promotes invasion of cancer cells in vivo and is a potential therapeutic target in some types of gastrointestinal cancers.

Three-dimensional reconstruction of the Ephrin-B2 female mouse model of anorectal malformations

Purpose Persistent cloaca is a rare, severe congenital anomaly. The Eph family receptor tyrosine kinases and their ligands, the ephrin family, mediate diverse embryonic cell-cell recognition events. We previously reported ephrin-B2lacZ/lacZ mice manifest male high imperforate anus and female persistent cloaca (Dravis et al. Dev Biol 2004; 271(2): 272-290). We studied and demonstrated via 3D-reconstruction whether altered ephrin-B2 signaling in female ephrin-B2 lacZ/lacZ mutant mice affect apoptosis, thereby possibly contributing to persistent cloaca malformation. Material and methods Female embryonic days 11.5, 13.5 and 16.5 (E11.5, E13.5 and E16.5) wild type (WT), ephrin-B2lacZ/+ (Z/+) and ephrin-B2 lacZ/lacZ (Z/Z) mice sections were dual labeled for ephrin-B2 and TUNEL immunohistochemistry. Anti-galactosidase antibody detected the ephrin-B2-gal fusion protein in Z/+ and Z/Z mice. WinSurf software was used for 3D reconstructions of the cloacal region. Results In WT E11.5 females, the cloacal membrane (CM) is intact. The urorectal septum (URS) is partially separating the cloaca into urogenital sinus and hindgut; no vagina has developed. In contrast, Z/Z females had poor cloacal development. Z/+ embryos showed widened URS. Apoptosis in WT females occurred prominently in CM, dorsal cloaca, and hindgut. In contrast, apoptosis in Z/Z females was limited to the endoderm of the poorly formed cloaca. These changes progressed throughout E13.5 and finally at E16.5, Z/Z females exhibited persistent cloaca. Conclusions The 3D reconstruction of the cloacal region provides a better understanding of the mechanism by which altered ephrin-B2 signaling yields persistent cloaca via diminished apoptosis in Z/Z mice.